| Apple cultivars that are susceptible to postharvest carbon dioxide can develop external carbon dioxide injury during storage in poorly ventilated conditions or in high carbon dioxide controlled atmosphere.The early symptom of the injury is slight browning on the surface of apple peels,which can develop to brown lesions with irregular shape.Bagging is a commonly used apple cultivation technique in China;however,production experience has found that failure to remove the bags before harvest significantly reduces the postharvest carbon dioxide tolerance of the peels of bagged apples.The mechanism of this phenomenon has not been established.In this study,debagged Fuji apples（double-layer paper bagged,and bags removed 30 d before harvest）and bagged Fuji apples（double-layer paper bagged,bags not removed before harvest）were used as the materials to verify the impact of preharvest bagging and debagging on the postharvest carbon dioxide tolerance of Fuji apples,and explore the mechanism of external carbon dioxide injury of apples through statistical analysis of carbon dioxide injury incidence,microscopic structure observation,transcriptome analysis,determination of physiological and biochemical indicators,exogenous ATP treatment,and VIGS experiments for verification.The main findings of this study are as follows:1.Preharvest debagging significantly improved the carbon dioxide tolerance of Fuji apple peels.External carbon dioxide injury symptoms appeared on debagged Fuji apples after at least 60 d under carbon dioxide stressed conditions,while the symptoms appeared on bagged Fuji apples on about 8 d under the same conditions.The difference in postharvest carbon dioxide tolerance was not due to the differences in tissue structure of the peels.2.The occurrence of external carbon dioxide injury of apples was not caused by changes in polyphenol oxidase（PPO）activity but was closely related to reactive oxygen species（ROS）accumulation and membrane oxidative damage.Under carbon dioxide stressed conditions,the PPO activities of both bagged and debagged Fuji apple peels decreased significantly,and there was no significant difference between them;the increase rates of superoxide anion,hydrogen peroxide,and malondialdehyde（MDA）content in bagged Fuji fruit peels were significantly higher than that in debagged Fuji.3.The occurrence of external carbon dioxide injury of bagged Fuji apples was closely related to the decline in the antioxidant capacity and energy metabolism level.Analysis of antioxidant system-related indicators of the apple peels revealed that the antioxidant capacity of the peels decreased with the development of carbon dioxide injury;the total phenolic and ascorbic acid contents of debagged Fuji fruit peels were significantly higher than that of bagged Fuji peels,but both were not the direct reasons for the occurrence of carbon dioxide damage in bagged Fuji fruit peel;the superoxide dismutase and peroxidase activities of the apple peels were not affected by carbon dioxide stress or bagging conditions,respectively;preharvest debagging and carbon dioxide stress significantly reduce the catalase activities of the peels.Analysis of energy metabolism-related indicators of the peels revealed that debagged Fuji apple peels had high energy reserves and also maintained at a high level under carbon dioxide stress;the energy level of bagged Fuji apple peels were initially low,and decreased rapidly with prolonged stress time;carbon dioxide stress significantly inhibited the activities of succinate dehydrogenase and Ca2+-ATPase,enhanced H+-ATPase activity,and had no significant effect on cytochrome C oxidase activity in apple peels.The inhibition of succinate dehydrogenase indicated the inhibition of tricarboxylic acid cycle.Exogenous ATP treatment significantly increased the carbon dioxide tolerance of apple peels,further confirming that the occurrence of carbon dioxide injury in apple peel was closely related to changes in energy level.4.Transcriptome analysis revealed that glycolytic metabolism was the main energy compensation pathway of apple peels under carbon dioxide stress.The decrease in glycolytic metabolism level of bagged Fuji apple peels may be the main reason for the occurrence of external carbon dioxide injury.Gene ontology enrichment analysis of differentially expressed genes（DEGs）of both bagged and debagged Fuji apple peels were mainly enriched in cellular process,metabolic process,membrane,membrane part,cell,cell part,organelle,binding and catalytic activity.KEGG（Kyoto encyclopedia of genes and genomes）enrichment analysis of DEGs showed that in the early stage of carbon dioxide stress,the only pathway significantly enriched in bagged Fuji apple peels was glycolytic metabolism,but when injury occurred,this pathway was not significantly enriched;under the same conditions,the glycolytic metabolism pathway was consistently significantly enriched in debagged Fuji apple peels.5.Pyruvate kinase（PK）was a crucial enzyme regulating the change of glycolytic metabolism in apple peels in response to postharvest carbon dioxide stress.In glycolytic pathway,hexokinase and phosphofructokinase activities of peels were not affected by carbon dioxide stress;PK activities of the peels,however,were influenced by both bagging conditions and storage conditions.PK activities of bagged Fuji apples under carbon dioxide stress exhibited an increase-to-decrease trend,and were significantly lower than that of the other experimental groups from 5 d,which was associated with the changes of energy levels;the pyruvate contents of bagged Fuji peels were significantly lower than other experimental groups since 5 d.In ethanol fermentation,carbon dioxide stress had no effect on pyruvate decarboxylase activities of the peels;on 8 d,the alcohol dehydrogenase activities of debagged Fuji apples（without injury）and bagged Fuji apples（with injury）did not differ significantly;the ethanol and acetaldehyde contents of debagged Fuji apple peels were higher than those of bagged Fuji apple peels.These results indicated that the occurrence of carbon dioxide injury in apple peels was influenced by the change of PK activity,but not directly related to hexokinase and phosphofructokinase activities and ethanol fermentation.Silencing of the Md PK gene led to reduced carbon dioxide tolerance in bagged Fuji apple peels compared with the empty vector control.The energy level and antioxidant capacity of the Md PK-silenced fruit were significantly lower than those of the control,while the levels of ROS and MDA contents were significantly higher than those of the control,which further demonstrated the critical role of PK in the occurrence of external carbon dioxide injury of bagged Fuji apples.The above research results indicate that the occurrence of external carbon dioxide injury in bagged Fuji apples was closely related to changes in PK activity.During the early stage of carbon dioxide stress,the increase in PK activity in the peels enhanced the glycolytic pathway,compensating for the energy deficit caused by the reduced metabolism of tricarboxylic acid cycle.With the prolongation of carbon dioxide stress,the decrease in PK activity led to a decrease in glycolytic metabolism level in the peels,weakening the energy compensation effect and resulting in energy deficit,accompanied by the decrease in antioxidant capacity and accumulation of ROS,resulting in oxidative damage to membrane,and ultimately leading to the injury. |
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